Production of monospecific antiserum to a cytosolic epoxide hydrolase from human liver.
A method for the purification to apparent homogeneity of cytosolic trans-stilbene oxide hydrolase from human liver is presented. The method employed ion exchange and gel filtration chromatography. From 50 g of human liver, 4.9 mg of homogenous enzyme protein was obtained. Although the enzyme had lost much of its catalytic activity during purification, it was nevertheless suitable for the preparation of antibodies to the enzyme. Only one immunogenic species was present in the antigen preparation, but some antibodies that were cross-reactive to sites on catalase were present in the antiserum. These catalase-specific antibodies were removed by immunoaffinity chromatography, and an IgG fraction that is monospecific to the cytosolic epoxide hydrolase was obtained. The usefulness of antibodies to this enzyme in immunoblotting experiments, following either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focussing, as well as in enzyme-linked immunosorbent assays, is demonstrated.
Qato
M K
MK
Department of Pharmacology, University of Illinois College of Medicine, Chicago 60612.
Reinmund
S G
SG
Guenthner
T M
TM
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000917:Antibody Formation; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology; D004851:Epoxide Hydrolases / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007106:Immune Sera / Q000032:analysis*; D008099:Liver / Q000201:enzymology*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D007106:Immune Sera; D004851:Epoxide Hydrolases; C048095:stilbene oxide hydrolase